Our goal is to elucidate the mechanism of initiation of transcription and translation in S. cerevisiae. We will focus on the iso-1-cytochrome c gene (CYC1) of the organism. A genetic approach will be taken that employs lacZ gene fusions as a key analytical tool. Mutations will be isolated affecting the level of expression of CYC1 - lacZ fused genes. The fusions will reside on small plasmids that can be moved back and forth between E. coli and yeast. These experiments will be directed at the following questions: 1) What DNA sequences comprise the CYC1 promoter (i.e. sites where RNA polymerase II and regulatory proteins interact)? 2) What yeast regulatory proteins play a role in CYC1 expression? 3) Can CYC1 expression be altered by integrating the gene at different locations in the yeast genome? 4) What features of yeast mRNA other than the initiator AUG are important in translation initiation? As a consequence of these studies, we will elaborate a system which can be used to obtain expression of cloned heterologous genes in yeast. We hope that this system will facilitate the production of proteins of clinical or biological importance that can not be obtained easily from their natural source or from bacterial expression systems.